Deionized corn steep liquor in production of tetracycline



United States Patent DEIONIZED CORN STEEP LIQUOR IN PRO- DUCTION OFTETRACYCLINE Pasquale Paul Minieri, Brooklyn, N. Y., and Melvin C.Fir-man, Princeton, and Herman Sokol, Fort Lee, N. J., assignors toAmerican Cyanamid Company, New York, N. Y., a corporation of Maine NoDrawing. Application January 12, 1955 Serial No. 481,494

6 Claims. (Cl. 195-116) The present invention relates to a new andtherapeutically useful broad-spectrum antibiotic product and to itsproduction by a fermentation and recovery process. This application is acontinuation-in-part of our co-pending application Serial No. 382,637,filed September 28, 1953, now U. S. Patent No. 2,734,018.

We have discovered that under proper process conditions (as exemplifiedin the following), including use of an organism of the speciesStreptomyces aureofaciens or a mutant of such an organism, there isproduced in a fermentation broth and its crude product a high concentration of an antibiotic substance different from those known before,and different in particular from the substance chlortetracyclineheretofore produced from organisms of that species; that under properprocess conditions (also exemplified below) this substance can be formedin the fermentation broth and its crude concentrate with no materialconcomitant formation of chlortetracycline, and there can be recovered anew product containing this antibiotic substance in high concentrationwith little or even no detectable content of other antibiotic substance;and further that this product has properties which mark it as a new anduseful therapeutic product likely to prove superior to knownbroad-spectrum antibiotics.

We first produced this substance in good yield and relatively highconcentration from a fermentation broth otherwise conventional butsubstantially free of chloride, using as the organism a strain of thespecies S. aureofacz'ens found in a soil sample obtained in Texas. Wehave since produced the substance in higher concentration using a mutantof that organism, designated UV-8, having distinctive characteristics;and we have reason to believe that further mutants of this or otherorganisms will permit production of this substance in high concentrationin a broth containing a substantial content of chloride.

The new antibiotic substance of the present invention is tetracycline.The name tetracycline was first applied to the molecular portion commonto aureomycin and terramycin (Brunings et al.; J. A. C. S., 74:4976-4977, October 5, 1952); and later to a theoretical substance ofwhich it was speculated that it might show antibiotic activity (Regna;Iour. N. Y. Acad. of Science, Ser. II, vol. 15, No. 1, pages 12-17,November 1952). Preparation of tetracycline by a catalytic hydrogenationof chlortetracycline has been reported recently (I. A. C. S., 75: 4621,4622, September 20, 1953). Prior to those publications or information oftheir contents we had produced tetracycline which we designatedsubstance HA- 20A and had recognized its relation to but difference fromeither of the substances now designated as chlortetracycline andoxytetracycline. Its freedom from chlorine differentiated it at oncefrom chlortetracycline, and its behavior in certain respects indicated adifference from oxytetracycline, which subsequent study has confirmed,despite a resemblance in other respects.

The antibiotic substance is an amphoteric substance which forms saltswith acids and bases. Its solubility in Proteus vulgaris Klebsiellapneumoniae Salmonella schottmillleri Mycobacterium tuberculosisStaphylococcus aureus Sarcina lutea Bacillus subtilis Escherichia coliAerobacter aerogenes Other bacteria are also affected in varying degreesas will bemore fully shown.

The acid salts of tetracycline base may be readily prepared bydissolving the free base in water or other suitable solvent acidifiedwith an organic or inorganic acid, for example, hydrochloric acid,sulfuric acid or acetic acid. Similarly the basic salts may be readilyformed by adding an organic or inorganic base, for example, sodiumhydroxide, calcium hydroxide, organic amines or quaternary ammoniumsalts to a solution of the free base.

Tetracycline can be distinguished from previously known antibiotics inmany ways. A chemical analysis of the free base shows that theantibiotic contains the following elements: Carbon, hydrogen, nitrogenand oxygen (by difference). Since it does not contain sulfur orchlorine, it is distinguished from many others and particularly fromchlortetracycline, which contains chlorine.

As is characteristic of many organic compounds, the properties ofcrystalline tetracycline are somewhat dependent upon the degree ofpurity and the manner in which the crystals are formed. Whenprecipitated from solutions containing water, the free base precipitatesas the trihydrate, which may be dried to the anhydrous form. Normally.the free base is used in the trihydrate form, as the anhydrous form isquite hygroscopic and hydrates readily in the presence of moisture.

Crystals of tetracycline free base trihydrate were examinedcrystallographically and were found to belong to the orthorhombicsystem. These crystals exhibited the following optical characteristics:

The optical rotation of highly pure crystalline tetracycline free basein methanol was determined.

(a) -=-234 (0.82% in methanol) I The precise solubility of the freebase, as is the case with most antibiotics, is closely related to thedegree of purity. In Table I are listed approximate solubilities of thefree base trihydrate of high purity in various organic solvents.

TABLE I Solvent Solubility in 100 ml. Methanol At least 3 g. Ethylacetate mg. chloroform 20 mg. Water Less than 10 mg. Benzene Less than10 mg Diethyl ether Less than 10 mg.

The melting point of anhydrous crystalline tetracycline 'free base, asfor many other organic materials, depends to a certain extent on anumber of factors including the degree of purity and the manner in whichthe crystals are heated. Crystalsof highly pure anhydrous free base on amicroscope slide were placed on a hot stage at 100 C. The temperaturewas increased at the rate of 2 per minute until a temperature of 145 wasreached and then at 1 per minute until the crystals melted. When heatedunder these conditions, the melting point was in the range of 160-l68 C.with decomposition. If the stage is heated to 155 C. initially and thetemperature of the sample is increased at the rate of 1 per minute, thesubstance melts over asomewhat narrower range. While these crystals werenot absolutely pure, it is believed that this range is characteristic.

A sample of the trihydrate in a capillary melting point tube was placedwith a thermometer in a test tube containing Dow-Corning Silicone No.200 at l-155 C. This test tube was immersed in a bath of the samematerial at l165 C., which was then heated with stirring so that thetemperature rose 1-2" per minute. The sample expanded slowly when theinner tube temperature rose to 160-165 C. There was no darkening but ayellow color developed. When the temperature reached 170-175 C.,darkening started to take place. This became more pronounced with afurther increase in temperature. When a sample of the anhydrous freebase was subjected to the same treatment, similar results were obtained.

The optical rotation of a solution of highly pure tetracyclinehydrochloride crystals in water was determined.

(00 -=283 (0.65% in water) The ultra-violet absorption of both the freebase and the hydrochloric acid salt of tetracyline was determined bymeasuring the optical densities of methanol solutions versus a methanolblank at various wave lengths (Beckman Squartz spectrophotometer-ModelDU). A solution of 1.0 mg. of the free base in 50 ml. of methanol gavethe following maximum and minimum values:

The ratio of Elf at 269 to that at 365 is 1.00 and is characteristic ofthe purified antibiotic.

A solution of 1.10 mg. of tetracycline hydrochloride in 50.0 ml. ofmethanol gave the following maxima and minima:

El? at 234 mu=179 Ell... at 270 mu=301 E11,, at 298.15 mu=131 The ratioof Flf at 270 to that at 363 is 1.10 and is characteristic of theantibiotic.

dredths:

7.3 microns 8 55 microns 10.6 microns 12.55 microns 7.5 microns 8.9nvcrons 11.3 microns 12.9 microns 7.8 microns 9.3 micons 11.7 microns13.85 microns 8.3 microns 9.65 microns 12.0 microns 14 microns 12.?microns 14.45 microns The infrared absorption spectrum of thehydrochloride salt in a neutral mineral oil mull is shown in Figure 2.The location of the characteristic absorption bands in thecharacteristic fingerprint region of the spectrum are as follows, allvalues being rounded to the nearest five hundredths:

5 microns llllClOllS llllCl'tJllS 10.1 microns 10.6 microns 11.7 microns12.75 microns 7.0 microns 8 7.5 microns 8. 7.7 microns 89 microns 7.9microns 9.75 microns Tetracycline, the antibiotic produced by theprocess of the present invention, is characterized in further detail inouraforesaid application, Serial No. 382,637. The physi cal, chemicaland physiological properties of the antibiotic have also been describedin numerous scientific publications and further enlargement upon theseproperties appears to be unnecessary at this time.

Tetracycline is produced by fermentation using a microorganismexemplified by one newly isolated from a soil sample collected in theState of Texas and by various organisms of the same species. It also isproduced by mutants, some of which have developed characteristics nottypical of the species. The Texas organism belongs to the speciesStreptomyces aureofaciens described in Dug gar Patent No.2,482,055-Aureomycin and Preparation of Same.

Table 11 sets forth the cultural characteristics of two isolates derivedfrom the Texas culture which have been used in the production oftetracycline. These characteristics .25 microns 129 microns 13.6 14.5

are identical with those exhibited by a known culcnre of S.aureofaciens.

TABLE II Glucose broth Yellowish growth settled on the bottom, acidreaction. Ycllowish growth settled on the bottom, no acid reaction.

Yellowish growth settled on the bottom, no acid reaction.

Sucrose broth Yellowish growth settled on the bottom. acid reaction.

Surface growth. slight clearing. no pH change.

Lactose broth Glycerin broth Litmus milk Gelatin stab Growth good at thesurface. no liquefaction, yellow pigment. Asparagine-meat extract agarSlight yellow pigment good growth. aerial hyphac,

white becoming grey.

Poor growth, flat colorless mycelium, no aerial hyphnc, no solublepigment.

Emerson agar Heavy growth, brownish mycelinm, no aerial hyphne. backbrownish slight solo ble brown pigment.

Same as Emerson.

Growth heavy, white ncriul hyphae turning grey, back greenish yellow.slight sol uble yellow pigment.

Flat colorless mycelium. no aerial hynhae no soluble pigment.hydrolysis.

Growth heavy. aerial hyphatwhite turning grey, pigs ment brownish.

Czapek-Dox agar Nutrient glucose agar Glycerin asparagme agar"--.

Starch agar n Calcium malate agar The strains, variations or mutants ofSlrcptomyces aureofaceins which are preferred are those which producerelatively high yields of tetracycline in the fermentation broth,particularly those which will produce more than 500 meg/ml. An exampleis an organism, UV-B, which is a mutant of the Texas microorganism. Thismutant is a new strain which produces such yields in the fermentationmedium and is a strain not described heretofore. Strain UV8, when grownon Waksman agar (Journal Bacteriology: 7: 339-341, 1922) exhibits heavymycelial growth which is cit-white at first, becoming yellow and whichis gradually covered with a powdery white aerial mycelium, which latersporulates. Old slants exhibit a jet-black coloration with small patchesof white mycelium. This black growth consists of a mass of bodies inshort chains which are easily disrupted. These bodies are variable insize and shape, ranging approximately from 0.5 up to 4.5 mu and varyingin shape from oval to slightly angular globose-spherical. The averagesize 'is somewhat greater than those in S. aureofacz'ens (NRRL2209) andthere is greater variationin size and shape.

The present invention is not limited to UV-8 or any particular organismbut includes any S. aureofaciens organism or variant or mutant, eithernaturally occurring or artificially induced, which produces tetracylinein concentrations making possible the recovery of the therapeuticproduct. in fact, the invention is not necssarily restricted to anyspecies of Streptomyces. Control of the chloride content of thefermentation medium is one of the critical parts of the invention andthe process is broadly applicable to any fermentation process utilizinga tetracycline producing microorganism which also tends to producechlortetracycline in the presence of chloride ions. Many diiferentstrains of streptomyces which have the ability to produce bothchortetracycline and tetracycline havebeen observed and while it isbelieved that all of these are properly classifiable as Streptomycesaureofaciens despite some rather marked diiferences in their growthhabits and nutritional requirements, note Duggat et al.: Annals of theNew York Academy of Sciences, volume 60, pp. 71-101, published October29, 1954, yet there are those who might be inclined, for one reason oranother, to assert that their tetracycline-producing microorganismbelongs to a different species. In view of this possible conflict ofterminology and in view of the generic nature of the present invention,applicants wish "to include within the scope of the claims herein theuse of all microorganisms having the tetracycline-producing qualitiesset forth above.

As stated in our earlier co-pending application, thefermentation mediumfor use in the process of the invention, contains sources of assimilablecarbon and nitrogen, inorganic salts, minor elements and various otheragents which will be mentioned in greater detail hereinafter. It is alsopointed out in our co-pending application that most strains of S.aureofaciens which produce tetracycline also tend to producechlortetracycline if chloride ions are available. Theoretically, onepart of available chloride ion can result in about 14 parts ofchlortetracycline. Since many S. aureofaceins are efficient utilizers ofchloride ions, it is desirable that the chloride ion be kept at aminimum. Ideally, the chloride ion should be even less than one part permillion. However, it is not economically feasible to producefermentation media having this low concentration of chloride ion andtherefore our invention contemplates the use of higher concentrations ofchloride ion in the fermentation medium.

The preparation of fermentation media containing less than parts permillion of chloride ion is comparatively easy when the medium is aso-called synthetic medium, that is, one in which the carbonrequirements of the fermentation are supplied by relatively purematerials such as sucrose and the nitrogen is supplied by ammoniumsulfate or other highly purified chemical substances. Unfortunatelythese synthetic media are very expensive and lack some of the essentialmetabolites of the fermentation process wherein high yields oftetracycline may be obtained. For this and other reasons not fullyunderstood, it is desirable to use naturally-occurring materials inpreparing fermentation media such as, for example, corn steep liquor,animal stick liquor, casein digests, and the like. Not only are thesematerials 6 cheaper than are the materials of the synthetic media, butthey result in much higher yields of the desired antibiotic.Unfortunately these materials carry substantial quantities of chlorideions and in producing tetracycline by fermentation, undesirably largeamounts of chlortetracycline are concomitantly produced.

Tetracycline is considered to be a superior antibiotic and physiciansgenerally wish to treat their patients with drugs of known composition.It is preferred, therefore, that the antibiotic be substantially pure.Limits on the purity of tetracycline offered for sale as such, have beenestablished so that commercial tetracycline should contain at least 96%tetracycline. Accordingly, it is highly desirable that the process ofproducing tetracycline yield a product which is essentially free ofother antibiotics. During the processing and purification operations,some of the chlortetracycline that may be contained in the fermentationbroth is eliminated by the isolation procedure and by chemicaldecomposition, particularly under alkaline conditions, and a crudefermentation liquor may serve as a starting material in which there maybe as much as about 8-10% chlortetracycline, without incurringunnecessary expense in separating the undesirable chlortetracyclinecomponent.

As noted in our co-pending application, Serial No. 382,637, naturalmaterials often used fermentation media contain chloride ions, but thesematerials may be used, nevertheless, if they are first deionized withion exchangers or other suitable means of removing chloride ions.Various ion exchange materials may be used for these purposes and theirnature and manner of use will be described in more detail hereinafter.

As shown in our co-pending application, natural materials such as cornsteep liquor may be diluted with water to a concentration suitable forpassing through an ion exchange bed and the chloride ion removed to theextent considered necessary. Additional nitrogen, carbon and mineralelement requirements of the fermentation medium may then be added to thede-ionized corn steep solution and the fermentation medium is then readyfor use in the process. Corn steep liquor contains substantialquantities of chloride ions, of the order of 0.15% to 0.5% by weight,and greater care must be exercised in treating material of this typethan in the case of other natural substances used in the preparation offermentation medium.

Casein, which is a very useful material for preparing fermentationliquors, is apt to contain large quantities of chloride ion dependingupon the method in which it was prepared. Amounts of chloride ion up to0.30% are frequently encountered. Enzymatic digests of casein which arealso useful in preparing fermentation media may contain even higherconcentrations of chloride ion and these products should be treated inaccordance with the teachings of the present invention, if used in thepreparation of fermentation liquors for the production of tetracycline.

Animal stick liquor, meat meal and fish meal digests and various othermaterials, particularly of animal origin, contain large quantities ofchloride ion and solutions prepared from these substances should betreated in ac cordance with the teachings of the present invention toreduce their chloride ion content.

It will be understood, of course, that components of the fermentationmedium which are in insoluble form cannot be passed through ion exchangebeds without soon stopping them with insoluble deposits. The process ofthe present invention contemplates treatment of soluble components ofthe medium such as those mentioned herein. Highly viscous substances arealso ditficult to pass through ion exchange beds-and must, therefore, bediluted with water to a consistency which will permit am'ple flowthrough the bed.

In some instances where insoluble materials are to be added to thefermentation medium, these may be washed materials in accordance withthe present invention.

"aseeg'r aa with deiorii'z'e'd wat'erto -'r'e'in'ov'e chloride -ionsprior to adding 'thern'to the fermentation medium. Obviously, the natureof the material willdetermine the treatment. Raw starch, for example,which often contains considerable quantities of chloride ion 'may bewashed with deionized water added to the fermentation medium and thencooked 'or otherwise treated to make it available for assimilation bythe fermenting microorganism. Soluble forms of starch may when properlydiluted be passed through the ion exchange bed to remove chloride ions.

Soy bean meal preparations often contain excessive amounts of chlorideions for the production of tetracycline and when materials of this typeare used in significant amounts, it should also be treated by ionexchange techniques as described above. The same observations apply toother naturally occurring materials which are used to preparefermentation liquors when their chloride ion content is too high.

In our co-pending application, Serial No. 382,637, we state that weprefer to use tetracycline producing strains of Streptomycesaureofaciens which will produce more than 500 micrograms per milliliterof the antibiotic. In such cases the fermentation medium may contain 10parts per million of chloride ion although lower amounts are preferred.Further, improvements in selecting strains of tetracycline producingstreptomyces have resulted in marked increases in the yield oftetracycline that can be formed so that it is now easily possible toproduce 5000 micrograms per milliliter, and even more, of tetracycline.This result is achieved not only by the selection of particularlyhigh-producing tetraycyline strains, but also by careful selection ofthe materials making up the fermentation medium and restriction of thechloride ion content. Our invention, therefore, contemplates the use ofstrains of S. aureofaciens capable of producing amounts of tetracyclinein excess of 500 micrograms per milliliter in fermentation mediacontaining natural materials of the type now commonly used in otherfermentation processes which have, however, been treated to eliminate asmuch of the chloride ion content as possible. In this manner, it ispossible to produce crude fermentation broths having only a smallproportion of the total antibiotic present as chlortetracycline.

As noted above, it is permissible, although not desirable, to have asmuch as 8 to 10% of the total antibiotic as chlortetracycline in thecrude fermentation broth. As will be seen, therefore, a fermentationbroth containing 5000 micrograms per milliliter of tetracycline mighthave as much as 500 micrograms per milliliter of chlortetracycline,which could result from the presence of 40 to 50 parts per million ofchloride ion in the fermentation media when the microorganism is anefficient utilizer. If the particular strain employed in the processdoes not fully utilize the chloride ions, the fermentation liquor maycontain larger amounts of chloride ion. In such instances many naturalmaterials need not be treated in accordance with the process of theinvention as their chloride ion content is already sufficiently low.However, the most effective substrates for the formation of high yieldsof antibiotic include corn steep liquor, which, as previously noted, isvery high in chloride content and fermentation liquors containingsignificant amounts of this material can be improved if treated. V

In common with other fermentation processes for the production ofantibiotics, the fermentation liquors will contain sources ofassimilable carbon, nitrogen and mineral salts. Many of these arerelatively free of chloride ions and are used to supplement thematerials referred to above which are treated with ion exchange Theseinclude such things as sucrose, glucose, dextrin, sugar alcohols, citricacid, starch,cotton seed meal, cornmeal, soy bean meal, peanut meal andvarious other carbonaceous and nitrogenous substances which are used invaryingamountsin from about A to 5% by weight or more of the totalweight of the fermentation medium. For purposes of higher yields andeconomy, mixtures of these materials are often used together with thenaturally occurring products referred to above, which have been treatedwith ion exchange materials to remove excess quantities of chlorideions.

Mineral salts are also commercially added to fermentation media invarying amounts to support the growth of the microorganism and topromote high yields of tetracycline. Among these may be mentionedammonium phosphate, potassium phosphate, magnesium sulfate, calciumcarbonate and various trace elements including cobalt, copper, zinc,manganese, iron, chromium and still others. The use of these traceelements in promoting the fermentation is well understood by those inthe art and further description appears to be unnecessary.

As will be apparent, treatment of aqueous solutions containing nutrientmaterials for the fermentation process for the removal of chloride ionsmay, by virtue of the non-selective anion-absorbing capacity of someanion exchange materials, tend to remove some of the desirable anionscontained in the fermentation liquor. For example, phosphate ions may beremoved from the solution under some conditions. Similarly, the sulfateanion may also be removed. To avoid this possibility and to reduce theload on the ion exchange column, it is preferred that most of themineral elements such as the phosphates and sulfates, as well as thetrace elements, be added subsequent to chloride removal by ion exchangetreatment.

The principal object of the ion exchange treatment is, of course, toremove excess chloride ions from the naturally occurring materials whichcontain excessive amounts. Under some conditions it may also bedesirable to remove certain cationic groups prior to the fermentation.Accordingly, mixed beds which remove both cations and anions may be usedif desired. The use and control of such mixed ion exchange beds is amatter within the skill of the art and need not be elaborated uponfurther.

The ion exchange materials contemplated by the present invention includethose water insoluble substances which have the property of adsorbing orotherwise removing chloride ions from aqueous solutions. They aregenerally synthetic resins having free amino groups, for instance,phenol-formaldehyde-polyamine condensates,melamine-gmanidine-formaldehyde resins and polyalkylene-polyamineresins. The exact chemical nature of these substances and the manner inwhich they adsorb the chloride ions is not fully understood. They may,however, be purchased from various manufacturers by specifying that theyare intended for the removal of chloride ions from aqueous solutions.Various types are available on the open market.

Although substances could be used in the present invention which wouldsimply adsorb chloride ions, these would not be commercially feasiblebecause they could not be reused. The term exchange" is used inconnection with the ion exchange materials of the present invention tomean a material which after adsorbing chloride ions from solution can bereactivated by removing chloride ions thus adsorbed and the resin usedover again in preparing further batches of fermentation liquor.

Some ion exchange beds can be operated so that only anions are removedfrom solution. In others there may be a replacement. For example, in anion exchange resin operating on the sulfate cycle chloride ions areremoved and sulfate ions are given off to replace them in solution.Although it is not usually necessary, these sulfate ions can be adsorbedby a second ion exchange bed operated in tandem with the first. The samemay be true with respect to other anions which may be involved. Forthese reasons it is important that the anionic requirements or for theproduction of inoculum for inoculating 2.5% of the liquid volume in thetank.

tinuous agitation and aeration. 'tol.5 volumes of free air per volume ofliquid medium of the nutrient media, especially phosphate, be adjustedfollowing ion exchange treatment.

Following treatment of some of the constituents of the fermentationmedia with ion exchange resins, the other necessary materials that areused in the fermentation process may then be added to the treatedliquid, the whole sterilized, and inoculated with tetracycline producingmicroorganisms. The fermentation is then conducted in the usual manner.

During fermentation, it is desirable to grow the organism undersubmerged conditions with suitable aeration and agitation, as, forexample, in a flask on a shaking machine or in a stirred fermentorequipped with a sparger for inducing a stream of air continuously. Thetemperature does not appear to be critical within the range of 25 -35C., although the range of 30-3.3 C. is preferable. The initial pH of themedium should be close to neutrality, although some of the antibiotic isproduced in media with initial pH values as low as 5.0 or as high as8.5.

Buffering agents such as calcium carbonate and salts of organic acidssuch as the citrates, acetates and lactates are useful in maintainingthe pH within the proper range. In addition, the organic acids may serveas sources of carbon in the metabolism of the microorganism. The use ofa defoaming agent is desirable in large scale fermentors, even though inthis fermentation, foaming is not aparticularly difiicult problem and isreadily controlled by the use of conventional defoaming'agents such asoctadecanol in lard oil or other suitable commercial defoamer.

Inoculurn for fermentation may be prepared from growth obtained onslants inoculated with the S. aureofaciens. A suitable medium for theslants is Waksman agar having the following composition:

G./l. Glucose 10 Peptone KH PO 1 MgSO .7H O 0.5 Agar 20 The slant growthmay be transferred to shaker flasks which may be used as small scalelaboratory fermentors larger fermentations.

For large scale tank fermentation, the slant growth is used to seed asuitable liquid medium in shaker flask which is shaken on areciprocating shaking machine at temperatures ranging from 26 C.35 C. Asecond shaker-flask stage is usually employedin conjunction withsubmerged tank fermentations in order to increase the volume of theshaken inoculum equal to 1.5% to The reaction of the shaker flask mediumis within the range of pH 6.5

' to 7.5 initially and, as growth occurs, a continuous drop in the pHvalue is observed with values as low as 3.8 being recorded. However, aphysiological age as indicated by a pH value of about 5.0 isv apparentlythe most favorable period for transferring the inoculum.

. The shaker inoculum, equal to 1.5% to 2.5% of the liquid volume in thetank, is aseptically transferred to the fermentor and grown for about 2to 3 days with con- Air rates equal to 0.5

may be used depending upon thesize of the fermentor. Foaming of thebroth can be controlled by the sterile ad- I dition of an antifoam agentsuch as lard oil containing 2% octadecanol.

In the recovery of tetracycline from the ferementation broth, it ispossible to employ conventional practices developed in connection withother antibiotics, such as the known solvent extractions, With orwithout carriers, or

adsorptions. Of known solvents, such as butanol, ethyl acetate andchloroform, the use of which involves subse- "qtient purification steps,butanol is best for the extraction of tetracycline. These knownprocedures are applicable with fair success in the case of fermentationbroths in which little antibiotic substance other than tetracycline isformed, where the objective is simply to effect a separation between thetotal antibiotic content and the inactive materials ordinarily classedas impurities. In the case where there is a substantial formation ofantibiotic substance other than tetracycline, as when the chloride ionlevel is not kept to a minimum and there is substantial formation ofchlortetracycline, these known methods are less elfective if it isdesired to recover the tetracycline in high concentration in theend-product, omitting other antibiotic substances. (We refer here toantibiotics as substances antagonistic to E. coli.)

We have devised a novel recovery method which is highly effective ineither case mentioned above, and highly etfective both from thestandpoint of recovering a product free of inactive impurities and fromthe standpoint of recovering a product free of other antibioticsubstances concomitantly formed in the broth. This improved methodyields a crystalline product of high purity from both standpoints. Thismethod is exemplified below. In its main features, it involves using aparticular group of quaternary ammonium salts selectively precipitatingtetracycline from the broth, at an alkaline pH (pH 8 to 11) as thequaternary ammonium salt of tetracycline. This salt, after filtration,is then slurried with a small amount of water and a relatively largeamount of chloroform, whereby the quaternary ammonium salt oftetracycline is dissolved in the chloroform phase of the slurry. Thewater and. chloroform phases are then preferably separated to remove anyimpurities dissolved in water. The tetracycline is then extracted withan aqueous acid solution at a pH of about 1 to 2.5, with formation ofthe acid salt of tetracycline which goes into solution in the waterphase, from which it precipitates as a crystalline product upon increaseofthe pH to the range from 3 to 7, precipitation starting at the lowervalue.

The quaternary ammonium salts that are particularly useful in thisrecovery method are alkyl trimethylammonium chlorides and dialkyldimethylammonium chlorides wherein the alkyl group contains from 8 to 18(inclusive) carbon atoms.

Example I A corn steep liquor medium was prepared in the followingmanner. Corn steep liquor of approximately 50% solids content (w./w.)was diluted with distilled water to give a final concentration of 2%.The resulting diluted material was passed through a column packed with30 liters of a mixture consisting of 2 volumes of Amberlite IR 45, asanionic exchange resin, and 1 volume of Amberlite IRC 50, a cationicexchange resin.

Collection of the efiluent did not start until the resistivity had risento 5,000 ohm-centimeters and continued until the resistivity had droppedbelow 3,000 ohm-centimeters. To 1,000 ml. of the deionized corn steepliquor solution there was added the following components for thepreparation of a medium suitable for the propagation of inoculum and forthe production of tetracycline to produce a medium containing about 17p. p. m. of chloride,

although a lower chloride content is preferred:

For the preparation of an inoculum, SO-milliliter volumes of the abovemedium were distributed into 250- -h'tilliliter Erlenmeyer flasks and400-milliliter volumes into 2-liter flasks. A dry loopful of-spores fromthe surface growth of the tetracycline organism grown on slantedsemi-solid medium was used to seed the 50- n1illiliter volumes whichwere shaken at 30 C. for 72 hours on a reciprocating shaking machine('82 strokes per minute with a 2% inch displacement). At the end of thistime 1.0 ml. of the resulting mycelial suspension was used to inoculate400 ml. of medium which was shaken for an additional 28 hours. Four400-ml. volumes were pooled aseptically and used to seed the fermentor.

The fermentation medium was prepared in 25-gallon fermentors as follows:The components, at the concentrations described above were dissolved in11.5 gallons of. the deionized corn steep liquor solution for a finaltotal volume of 15.8 gallons. The medium was sterilized at 121 C. byinjecting steam at 15 p. s. i. gauge directly into the batch for 30minutes. The volume gained from the steam condensate provided therequired difference between the initial 11.5 gallons of deionized cornsteep liquor-sugar-salts solution and of the final 15.8 gallonsrequired. Upon cooling to 30 C., the mixture was seeded with the fourpooled 400-ml. volumes of inoculum previ ously described, agitated andaerated at a rate of 1.6 volumes of air per volume of medium for 38hours. At this time the reaction of the broth which was initially at pH5.9 had dropped to 4.1. The broth assayed 20 meg/ml. of antibiotic,using oxytetracycline as a standard and contained a substantial amountof tetracycline.

Corn steep liquor solutions at 1.5 and 2.0% concentrations were dionizedwith resin mixtures containing 3 volumes of anionic Amberlite resin IR45 and 1 volume of cationic Amberlite resin IRC 50 (ca. runs). One runwas made with Rohm and Haas mixed resin MB-l (2 volumes anionic resinIRA 400 plus 1 volume cationic resin IR 120). The ratio of resin volumeto efiluent volume of 1:3 was found satisfactory. A flow rate of 0.01ml. solution/ml. resin/minute gave better deionization than 0.016 ml.solution/ml. resin/minute. The use of two resin columns in serieseffected good deionization. Chloride ion assay of the solutionsdeionized in this way showed the presence of from 3.5 to 29 p. p. m.

Example 11 Corn steep liquor of approximately 50% solids content (w./w.) was diluted with distilled water to give a final concentration of1.5%. This diluted material was passed through a column packed with amixture consisting of three volumes of Amberlite IR 45, an anionicexchange resin, and one volume of Amberlite IRC 50, a cationic exchangeresin. Collection of the efiluent did not start until the resistivityhad risen to 5000 ohm-centimeters and continued until the resistivityhad dropped below 3000 ohm-centimeters. The following materials wereadded to the deionized corn steep liquor solution for the preparation ofa medium suitable for the propagation of inoculum and the production oftetracycline:

Sucrose percent 2.0 KH 'PO do 1.5 (NI-IQgHPQ; -4 e do 0.5 MgSO .7H O do0.2 CaCO do 0.1 ZnSO .7H O p. p. m 50.0 CuSO .5H O p. p. m 3.0 MnSO .4HO p. p. m 2.5 KBr percent .05

For use in preparation of both inoculum and fermentation flasks, thismedium was distributed in 100 ml. aliquots into 500 ml. Erlenmeyerflasks and sterilized by autoclaving for 20 minutes at 121 C., 15 p, s.i. The chloride content of the medium after sterilization was 17.7 p. p.m.

inoculum was prepared by seeding flasks witha lyophilized mixture ofspores and mycelium of Streptomyces rained the bulk of the activity,

aureofacins (strain 201-45) and incubating the flasks at 26 C. for 48hours on a reciprocating shaking machine. Five percent volumes of the 48hour inoculum was used to seed the fermentation flasks which were thenincubated under the same conditions for 43 hours. Antibiotic activitywas demonstrated by saturating a 13 mm. paper disc with filtered broth,placing the disc on an agar plate seeded with Escherichia coli andincubating at 30 C. for 18 hours.

Four hundred milliliters of this mash were treated with 2.4 g. of oxalicacid and 1.6 g. of ammonium oxalate. After stirring 10 minutes themixture was filtered. The filtrate was then stirred vwith ml. of ethylacetate and the pH adjusted to 8.5. The solvent phase, which conwasshaken with 5 ml. of water and the pH adjusted to 2. When diluted ,6with lower phase of system butanol-water containing 2.5% acetic acidabsorption maxima in the ultraviolet at 270 m; and 360 m were obtained.

Countercurrent distribution analysis (48 transfers, butanol-2.5% aceticacid) of this concentrate showed it to contain tetracycline. This wasaccomplished at the end of the distribution by dilution of each of thelower phases with methanol and measuring the optical density at 370 me.The bulk of the activity was found at tube 19. The peak tube numbers forchlortetracycline and oxytetracycline are 26 and 16, respectively.

Example III A shaker flask fermentation was conducted identical in allrespects to that described above, except that the anionic and cationicresins employed were Amberlite IRA 400 (2 volumes) and Amberlite IR (1volume), respectively. The medium contained 13.3 p. p. m. chloride aftersterilization. Antibiotic activity was demonstrated in the mash by thepaper disc-E. coli plate method.

When this mash was treated to concentrate the antibiotic activity asdescribed in Example ll, above, ultra violet absorption andcountercurrent distribution data were shown to be substantially the sameas those for the medium prepared with Amberlite resins RC 50 and IR 45.

Example 1V For the preparation of inoculum to be used in 25 gallonfermentors, 50 ml. volumes of the medium described in Example II weredistributed into 250 ml. Erlenmeyer flasks, and 400 ml. volumes into2-liter flasks. A dry loopful of spores from the surface growth of S.auerofaciens (strain T-5) on slanted semi-solid medium was used to seedthe 50 ml. volumes, which were shaken at 30 C. for 72 hours on areciprocating shaking machine. At the end of this time, 1.0 ml. of theresulting mycelial suspension was used to inoculate 400 ml. of medium,which was shaken for 28 hours. Four 400ml. volumes were pooledaseptically and used to seed the fermentor.

In preparing the fermentation medium, the corn steep liquor solution wasmade up in 2% concentration and deionized with the 3:1 mixture ofAmberlite resins IRC 50 and IR 45. The other components of the mediumdescribed in Example II, were dissolved in this deionized solution insuch quantities that, after 30 minutes sterilization at 121 C. byinjecting steam at 15 p. s. i. directly into the batch, the volumegained from the steam condensate provided a total volume of 15.8 gallonsof medium per 25 gallon fermentor.

After cooling to 30 C., the ferment ors, were seeded with 1600 ml.volumes of inoculum, mechanically agitated at -165 R. P. M., and aeratedat a rate of 1.6 volumes of air per volume of medium for 38 hours. Thebroths from two such fermentations contained 81 and 116 y/n'tl.

of antibiotic, respectively, when assayed with S. lu'teausingoxytetracycline as a standard.

, Ninety-six liters of mash, obtained by pooling "the above-describedbatches, were treated with 920 g. of oxalic acid and the pH adjusted to3.5. Hyfio was added, the

mixture-filtered and the filter cake washed with 4 liters of water. Thecombined filtrate and wash was extracted at pH 8.5 with 20 liters ofethyl acetatecontaining 1 liter of Emulsept (N- [lauroyl colaminoformyl-methyl] pyridine). The solvent extract was then stirred at pH 2with four successive 140 ml. portions of water. These aqueousconcentrates were pooled and adjusted to pH 7.8. The solids whichseparated were filtered, washed with water and then dried in a vacuumdesiccator. There were obtained 4.19 g. assaying 904 'y/mg. Thisrepresents an activity yield of 41%. The ultraviolet absorption ofthismaterial in 0.1 N sulfuric acid was determined. Maxima were found at 265mu and 360 mu with minima "14 rate of about 0.5 volume bfair per volumeof medium perminute; v

On harvesting, the fermented broth was found to contain over 5000micrograms permilliliter of tetracycline with less than 5% of thatamount of chlortetracycline. To regenerate the column, 1 N sulfuric acidwas passed through the column at the rate of 100 l./hr. until theeflluent contained no chlorideion (200 liters of acid required). Waterwas passed through at the rate of 100 l./hr. until the pH rose above2.0. water were required. The column is now ready for reuse. This columnhas been re-used fifteen times without at 232 mu and 301 mu. Fivemilligrams were distributed between chloroform and pH 7 phosphatebuffer. Substantially all the activity was found in thelower phase.Under these conditions, oxytetracycline concentrates in the upper phase.

Fifty milligrams of the product was dissolved in the least amount of Nhydrochloric acid and the solution seeded with a few crystals ofchlortetracycline and the mixture was allowed to stand at roomtemperature. Crystallization began within 15 minutes. The mixture wasplaced in the icebox overnight. The next day the crystalline solids wereseparated and dried on a porous porcelain plate. An ultravioletadsorption spectrum of the crystals in 0.1 N sulfuric acid solutionshowed that maxima were present at 267 mu and 360 mu and minima at 234mu and 301 mu. A solution of the crystals in concentrated sulfuric acidcontaining boric acid gave a red fviolet coloration. Oxytetracycliue andchlortetracycline under analogous conditions gave cherry'red and purplecolorations, respectively.

Example V 17.8 kg. of Amberlite IR 4B, a weakly anionic exv change resinof the phenol-formaldehyde-polyamine type sold by Rohm and Haas, wasneutralized with 1 N sulfuric acid, packed into a six inch column to aheight of 76 inches, and washed with water to an effluent pH of 2.0.

15.0 kg. of corn steep liquor were dilutedwith145 liters of water andfiltered at 50 C. The cake was washed with 15 liters of water. The cornsteep solution was passed through the column at the rate of 40 litersper hour. A positive chloride test was obtained in the eluate after theequivalent of 13.5 kg. of steep had been treated. The chloride-freetreated steep was adjusted to a pH of 4.0 with concentrated ammoniumhydroxide and was then ready for use.

A fermentation medium was made up with the following components:

Tap water to 100 ml.

The medium was sterilized 15 minutes at 125 C. and then diluted with 3vols. of tap water. This medium contained approximately 24 parts permillion of chloride ion. It was then inoculated with 2% of its volumewith S. aureofaciens strain S-77 containing 1% by weight in an inoculumsolids basis'corn steep liquor, 3% dextrin,

diminution of its chloride-removing capacity or diminution in thenutritional character of the resulting corn steep liquor.

Example VI A column similar to that of Example V was prepared 'usingIRA-400, a strongly anionic adsorbing ion exchange resin of thephenol-formaldehyde-polyamine type produced by Rohm and Haas. Corn steepliquor was passed through the column as in Example V and a fermentationmedium prepared, sterilized and inoculated with the same tetracyclineproducing strain of S. aureofaciens'as before, and the fermentationconducted in a similar manner. This particular fermentation mediumcontained approximately 18 parts per million of chloride ions. 1

On harvesting the broth, it was found to contain 3510 gammas permilliliter of tetracycline and 255 gammas per milliliter ofchlortetracycline.

Example VII A mixed cation-anion exchange resin bed was prepared withequal parts of IRA-400 and IR-IZO, the latterya strong cation exchangingresin of the sulfonic acid :type sold by Rohm and'Haas, and treated with1 normal ammonium sulfate similarly to the manner shown in preparing thecolumn in Example V. Corn steep liquor was treated as described above toremove chloride vions with this column and a fermentation mediumprepared in the same manner as shown in Example V. Theadiluted andsterilized medium .was then inoculated with S. aureofaciens strain S-77and the fermentation conducted under the conditions described in ExampleV. The fermentation medium at the beginning of the fermentationcontained approximately 23 parts per million of chloride ion. Thefermented broth Was found to contain 4670 gammas per milliliter oftetracycline and 390 gammas per milliliter of chlortetracycline.

Example VIII Corn steep liquor was diluted with water and treated withsilver nitrate to remove chloride ions as silver chloride. The chloridefree steep liquor was incorporated into a fermentation similar to thatof Example VII and inoculated with S. aureofaciens, and the fermentationconducted as before. On analysis, the fermented broth was found tocontain 3.600 gammas per milliliter of tetracycline and 270 gammas permilliliter of chlortetracycline.

Example IX In shaker fiask experiments, using the fermentation media ofExample V in which the medium was enriched with 200 milligrams per literof phosphoric acid, there 7 was obtained 2830 gammas per milliliter oftetracycline 0.625% calcium carbonate, and 0.2% ammonium sulfate.

and 210 gammas per milliliter of chlortetracycline.

When the fermentation is conducted as in the preceding paragraph, usinguntreated corn steep liquor at the rate of 1% total solids, thefermented broth is found to contain approximately 1600 gammas permilliliter of tetracycline and 1600 gammas per milliliter of chlortetracycline, thus showing the utilization of the available chloride ion inthe corn steep liquor to produce higher proportions ofchlortetracycline.

450 liters of i 15 Example X The anionic exchange resin may beoperated-in various forms such as the sulfate,nitrate, hydroxyl and thelike. when the process described in Example V was repeated with the ionexchange resin on the nitrate cycle, being prepared by treatment with 1normal nitric acid instead of sulfuricacid, two different runs gave 3590gammas of tetracycline per milliliter and210 gammas of chlortetracyclineper milliliter in one run, and 4460 gammas of tetracycline permilliliter and 230 gammas of chlortetracycline per milliliter in thesecond run.

In the foregoing runs the fermentation liquor contained from 15 to about30 parts per million of chloride ion and was fully utilized bythe S.aurebfaciens microorganism to produce all of the chlortetracycline thatwas possible in view of the chloride content of the medium. Be-

cause of the high yields of antibiotic thatwere obtained,

However, it is noted that the proportion of chlortetracycline in theseruns, wherein ion exchange resins have been used to treat corn steepliquor, is less than 8% in all cases.

We claim:

1. In a process of producing tetracycline;by aerobic fermentation with atetracycline-producing microorganism of the .Streptomyces genus which isalso capable :of

producing chlortetracycline in the presence of chloride ions, theimprovement which comprises contacting an aqueous solution of the watersoluble components of the fermentation medium with an ion exchangematerial to remove chloride ions therefrom.

2. The method of producing tetracycline which comprises the steps ofpreparing an aqueous nutrient medium, contacting said aqueous mediumwith an anionic ion exchange material to remove chloride ions from-saidsolution and thereafter inoculating said aqueous nutrient medium with atetracycline-producing strain of Streptoinyces aureofaciens and allowingaerobic fermentation to take place until tetracycline is produced.

3. In a process of producing tetracycline byaerobic fermentation of anaqueousnutrientmedium with a chloride-utilizing tetracycline-producingstrain of Streptomyces aureofaciens, the step which comprises subjectingan aqueous solution of corn'steep liquor to the action of an anionicexchange resin to reduce the chloride content thereof, adding saidtreated corn steep liquor to the fermentation medium, and allowing theS. aureofaciens to ferment saidmedium and produce tetracycline.

t 4. A process in accordance with claim 3 in which the total chloridecontent of the medium is between the range of 10 to parts per million.

5. A method of producing tetracycline which comprises the steps ofpreparing an aqueous nutrient medium, contacting said aqueous mediumwith an anionic exchange resin to reduce the chloride content thereof toless than about 50 parts per million, and thereafter in oculating saidaqueous nutrient medium with a tetracycline-producing strain of S.aureofaciens and allowing aerobic fermentation to take place untiltetracycline is produced therein in amounts of at least 500 parts permillion and is the major antibiotic substance therein.

6. A process in accordance with claim 5 which includes the step of.adding to said deionized aqueous nutrient medium a quantity of phosphateions.

References Cited in the tile of this patent UNITED STATES PATENTSAntibiotics and Chemotherapy, vol. IV, pp. 375-379, 450 452.

Martin et a1.: 2nd Am. Symposium on Antibiotics,

October 1954, U. s. D. H. E. w., Paper No. 156, Ab-

stract in Div. 63.

Nachod: Ion Exchange, 1949, Academic Press, N. Y., pp. 132, 145, 135,136, 374, 375.

1. IN A PROCESS OF PRODUCING TETRACYCLINE BY AEROBIC FERMENTATION WITH ATETRACYCLINE-PRODUCING MICROORGANISM OF THE STEREPTOMYCES GENUS WHICH ISALSO CAPABLE OF PRODUCING CHLORTETRACYCLINE IN THE PRESENCE OF CHLORIDEIONS, THE IMPROVEMENT WHICH COMPRISES CONTACTING AN AQUEOUS SOLUTION OFTHE WATER SOLUBLE COMPONENTS OF THE FERMENTATION MEDIUM WITH AN IONEXCHANGE MATERIAL TO REMOVE CHLORIDE IONS THEREFROM.